| Background:Adoptive transfer of activated T and NK cells has had significant clinical benefits in certain tumor models. Cytokine induced killer (CIK) cells are a group of cells that possess both T and NK cell like recognition of target cells. These cells are generated after extensive ex vivo manipulation of PBMCs. Maintenance of not only CIK cells but other activated effector T and NK cells in culture is vital for their effective transfer and development following adoptive immunotherapy. IL-21is the newest member of the common y chain family which has been shown to increase cytotoxic factors and cytokine secretion in immune cells without over stimulation. Such qualities make IL-21a suitable agent in immunotherapy of tumors. IL-21has shown effective antitumor function and is currently going clinical trials for tumors such as renal cell carcinoma, melanoma and lymphoma. Our previous experiment showed that like in T cells and NK cells, IL-21significantly improves the cytotoxicity of CIK cell on K562cells and primary leukemic cells from patients. Although proliferation of cells in a CIK cell pool was not observed we found that it helped maintain and grow the CD3+and CD56+phenotype. Our present experiment aims to explain the mechanism through which IL-21promotes CIK cells survival and cell cytotoxicity.Materials/Method:In our experiment, blood from healthy donors was collected and PBMCs were transformed into CIK cells following14days of culture using appropriate methods. The cells were then stimulated with IL-21for a defined period of time and subjected to MTT assays to measure cellular viability and cytotoxicity to K562cells. To elucidate the mechanism of action of IL-21, CIK cells were checked for the level of mRNA expression of perforin, Granzyme B, FasL, INF-y, TNF-a,Granzyme A,NKG2D, TNF-β using RT-PCR. Furthermore the expression of significantly important cytotoxic factors and cytokines was measured through flow cytometry and ELISA. Western blot was performed to check the involvement of JAK/STAT pathway following stimulation. Result:We found that IL-21doesn’t enhance in vitro proliferation of CIK cells, but does increase the number of cells expressing the CD3+/CD56+phenotype. IL-21can also significantly increase the cytotoxic potential of CIK cells to K562cells. It does so with significantly increased production of perforin which increased almost2folds from (0.7592±0.1457) to (0.9831±0.1265); Granzyme B also by almost2folds from (0.4768±0.1589) to (0.7319±0.1639) and FasL which increased by almost2folds from (0.4608±0.2842) to (0.7381±0.2568). Increase in secretion of cytokines such as INF-y was observed from (25.8±6.1)ng/L to (56.0±2.3)ng/L; and TNF-a from (5.64±0.61)ug/L to (15.14±0.93)ug/L while no significant difference was observed in the expression of Granzyme A,TNF-β and NKG2D. Measurement of IL-21R receptor on CIK cell surface following IL-21stimulation caused a more than two folds increase in expression of IL-21R from1.88%to4.25%. We further affirm that JAK/STAT is actively involved in IL-21signalling. STAT3and STAT5b could be potential signalling mechanisms taking part in IL-21enhanced cytotoxic potential of CIK cells.Conclusion:Using this information we have concluded that increased expression of perform, Granzyme B, FasL, IFN-y and TNF-a plays a significant role in IL-21enhanced cytotoxic potential of CIK cells and STAT-3and STAT-5b signaling pathway are involved in the processes. Our data indicate that IL-21is a potent enhancer of antitumor function of CIK cells. As CIK cells and IL-21have both been shown to increase patient survival or tumor free periods in certain hematological malignancies using them in conjunction might be therapeutically more beneficial. |
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